Downloaded from rnajournal.cshlp.org on June 24, 2011 - Published by Cold Spring Harbor Laboratory Press Functional characterization of the conserved amino acids in Pop1p, the largest common protein subunit of yeast RNases P and MRP Shaohua Xiao, John Hsieh, Rebecca L. Nugent, et al. RNA 2006 12: 1023-1037 Access the most recent version at doi:10.1261/rna.23206 References This article cites 55 articles, 30 of which can be accessed free at: http://rnajournal.cshlp.org/content/12/6/1023.full.html#ref-list-1 Article cited in: http://rnajournal.cshlp.org/content/12/6/1023.full.html#related-urls Email alerting Receive free email alerts when new articles cite this article - sign up in the box at the service top right corner of the article or click here To subscribe to RNA go to: http://rnajournal.cshlp.org/subscriptions Copyright © 2006 RNA Society JOBNAME: RNA 12#6 2006 PAGE: 1 OUTPUT: Tuesday May 9 09:05:45 2006 csh/RNA/111795/rna232 Downloaded from rnajournal.cshlp.org on June 24, 2011 - Published by Cold Spring Harbor Laboratory Press Functional characterization of the conserved amino acids in Pop1p, the largest common protein subunit of yeast RNases P and MRP SHAOHUA XIAO,1 JOHN HSIEH,2 REBECCA L. NUGENT,1 DANIEL J. COUGHLIN,1 CAROL A. FIERKE,1,2 and DAVID R. ENGELKE1 1Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606, USA 2Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606, USA ABSTRACT RNase P and RNase MRP are ribonucleoprotein enzymes required for 59-end maturation of precursor tRNAs (pre-tRNAs) and processing of precursor ribosomal RNAs, respectively. In yeast, RNase P and MRP holoenzymes have eight protein subunits in common, with Pop1p being the largest at >100 kDa. Little is known about the functions of Pop1p, beyond the fact that it binds specifically to the RNase P RNA subunit, RPR1 RNA. In this study, we refined the previous Pop1 phylogenetic sequence alignment and found four conserved regions. Highly conserved amino acids in yeast Pop1p were mutagenized by randomization and conditionally defective mutations were obtained. Effects of the Pop1p mutations on pre-tRNA processing, pre-rRNA processing, and stability of the RNA subunits of RNase P and MRP were examined. In most cases, functional defects in RNase P and RNase MRP in vivo were consistent with assembly defects of the holoenzymes, although moderate kinetic defects in RNase P were also observed. Most mutations affected both pre-tRNA and pre-rRNA processing, but a few mutations preferentially interfered with only RNase P or only RNase MRP. In addition, one temperature-sensitive mutation had no effect on either tRNA or rRNA processing, consistent with an additional role for RNase P, RNase MRP, or Pop1p in some other form. This study shows that the Pop1p subunit plays multiple roles in the assembly and function of of RNases P and MRP, and that the functions can be differentiated through the mutations in conserved residues. Keywords: site-directed random mutagenesis; pre-tRNA processing; 5.8S rRNA processing; ribonucleoprotein complex; steady-state activity INTRODUCTION cleavage at high salt concentrations in vitro, and therefore is an RNA enzyme or ‘‘ribozyme’’ (Guerrier-Takada et al. Ribonuclease P (RNase P) is a ubiquitous endonuclease 1983). Eukaryotic RNase P has a very similar RNA moiety, that removes 59 leader sequences from precursor tRNAs but the protein complement is far more complex than that (pre-tRNAs). RNase P activity has been found in Bacteria, of the bacterial enzyme. Nine- and 10-protein subunits Archaea, Eukarya, and organelles (Frank and Pace 1998; have been identified in the nuclear RNase P from yeast and Xiao et al. 2002) and most forms of RNase P are ribo- humans, respectively (Chamberlain et al. 1998; Jarrous nucleoprotein complexes, containing both protein and 2002). The eukaryotic RNase P RNA appears to have lost RNA subunits. Bacterial RNase P consists of an essential the ability to cleave pre-tRNAs in the absence of the protein RNA subunit (z130 kDa) and a small, basic protein subunits. subunit (z15 kDa) (Frank and Pace 1998). Although both In the yeast Saccharomyces cerevisiae, nuclear RNase P subunits are required for the in vivo RNase P function, contains one RNA subunit, RPR1 RNA, and nine protein the bacterial RNA subunit alone can catalyze pre-tRNA subunits: Pop1p, Pop3p, Pop4p, Pop5p, Pop6p, Pop7p, Pop8p, Rpp1p, and Rpr2p (Lee et al. 1991b; Chamberlain et al. 1998). All 10 subunits are essential for yeast growth Reprint requests to: David R. Engelke, Department of Biological and the in vivo RNase P activity (Chamberlain et al. 1998 Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606, and references therein). RPR1 RNA is synthesized as a pre- USA; e-mail: [email protected]; fax: (734) 763-7799. Article published online ahead of print. Article and publication date are cursor (pre-RPR1 RNA) that contains an 84-nucleotide at http://www.rnajournal.org/cgi/doi/10.1261/rna.23206. (nt) 59-leader sequence and a 39-trailing sequence flanking RNA (2006), 12:1023–1037. Published by Cold Spring Harbor Laboratory Press. Copyright Ó 2006 RNA Society. 1023 rna232 Xiao et al. ARTICLE RA JOBNAME: RNA 12#6 2006 PAGE: 2 OUTPUT: Tuesday May 9 09:05:48 2006 csh/RNA/111795/rna232 Downloaded from rnajournal.cshlp.org on June 24, 2011 - Published by Cold Spring Harbor Laboratory Press Xiao et al. the mature RPR1 sequence (Lee et al. 1991a). Pre-RPR1 MRP (Lygerou et al. 1994; Srisawat et al. 2002), and is RNA is assembled into a ribonucleoprotein complex with conserved from yeast to human (Lygerou et al. 1996b). at least seven of the nine RNase P protein subunits before Attempts to characterize the function of Pop1p in vitro being processed to the mature form (Srisawat et al. 2002). have not been successful, largely because the individually Eukaryotic RNase P is closely related to another ribonu- expressed protein is not soluble and the yeast holoenzyme cleoprotein enzyme, RNase MRP, which has only been has not been reconstituted in vitro. Therefore, we muta- found in eukaryotes and appears to have evolved from genized the evolutionarily conserved regions of Pop1p and RNase P to perform specialized functions. The demon- examined the effects of the mutations on the in vivo strated nuclear function of yeast RNase MRP is pre-rRNA functions and biogenesis of RNase P and RNase MRP. in the maturation pathway of 5.8S rRNA (Lindahl and Alignment of the Pop1p sequences from yeast, worm, and Zengel 1995; Reilly and Schmitt 1995; Tollervey 1995). human previously revealed three highly conserved regions Recently, an additional role for RNase MRP in cell cycle (Lygerou et al. 1996b). In this study, we have found four progression in yeast through selective mRNA turnover has conserved regions in the Pop1p family, including the three been reported (Gill et al. 2004). The yeast RNase MRP regions identified previously, by amino acid sequence holoenzyme consists of one RNA subunit, NME1 RNA, and alignment of additional putative Pop1p homologs. Mutated at least 10 protein subunits (Schmitt and Clayton 1994; POP1 gene libraries were then created, in which highly Chamberlain et al. 1998; Salinas et al. 2005). Eight proteins conserved amino acids in the four conserved regions were are the same as those in yeast RNase P: Pop1p, Pop3p, randomized. Selections for viable sequence variations in Pop4p, Pop5p, Pop6p, Pop7p, Pop8p, and Rpp1p POP1 were followed by screens for conditionally defective (Chamberlain et al. 1998 and references therein). The mutations. Subsequent characterization of the partially protein subunit unique to RNase P is the Rpr2p, whereas defective POP1 mutations showed that most defective Snm1p and Rmp1p are specific to RNase MRP (Schmitt mutations affect the assembly and in vivo functions of and Clayton 1994; Chamberlain et al. 1998; Salinas et al. RNases P and MRP to various degrees. A few mutations 2005). NME1 RNA and the RPR1 RNA conform to similar that impair only RNase P function or only RNase MRP secondary structures and have several small patches of con- function were obtained, suggesting that Pop1p might served sequence found in all RNase P RNAs (Forster and participate differentially in the two enzymes. The collection Altman 1990; Frank et al. 2000; Li et al. 2002). Thus, RNase of Pop1p mutations with distinct phenotypes is anticipated MRP arose from eukaryotic nuclear RNase P through to be useful to further understand the biology of RNase sequence divergence of a duplicated RNA subunit gene P/MRP and RNA processing in the cell. and substitution of one protein subunit with two others. Functions of the individual protein subunits of yeast RESULTS RNase P and RNase MRP are largely unknown, although several protein subunits can bind RNA. For example, Alignment of Pop1p and homologs Pop1p and Pop4p bind directly to the RPR1 RNA (Ziehler et al. 2001; Houser-Scott et al. 2002), whereas Snm1p can To identify putative homologs of the yeast Pop1p from bind to the NME1 RNA directly (Schmitt and Clayton other eukaryotes, we performed a BLAST search of the 1994). A separate study reported that Pop3p could bind to NCBI database with the sequence of Pop1p (http:// the RPR1 RNA, pre-tRNA, and single-stranded RNAs www.ncbi.nlm.nih.gov/BLAST). The search revealed puta- (Brusca et al. 2001), although the specificity of these tive Pop1p homologs from Drosophila (NCBI accession no. interactions is not known. Recently, crystal structures and NP_572236), Anopheles (NCBI accession no. XP_307517), NMR structures of the archaeal homologs of Pop4p, mouse (NCBI accession no. NP_080616), and Schizosac- Rpp1p, and Rpr2p have been solved (Boomershine et al. charomyces pombe (NCBI accession no.